Pneumocystis carinii (Pc) is an opportunistic pathogen and a major cause of morbidity and mortality in AIDS patients. Current knowledge of and research on Pc physiology and pathogenesis is impeded by the lack of a consistent and continuous source of the parasite. Development of an axenic long-term culture system will make organisms readily available for all research on Pc, including development of more rational approaches to chemotherapy. The purpose of this subgrant is to provide comparative ultrastructural and immunocytochemical analysis of Pc parasites in rat- infected lung, fresh isolates from lung, and from axenic cultures. The purity, morphologic integrity, and distribution of life-cycle stages of Pc isolated by various methods will be compared. These factors will then be monitored by EM over time in axenic cultures. Preservation methods both for morphology and for antigenicity will be optimized for each life cycle stage. Information gained from these EM studies will help improve methods of isolation and of culture as well as add to the currently incomplete understanding of the life cycle and intracellular organization of Pneumocystis. Using available polyclonal and monoclonal antibodies, surface components on cultured Pc will be identified and compared with rat lung Pc by immunoelectron microscopy and EM cytochemistry. Carbohydrate surface components will also be characterized with lectin-gold conjugates. It is critical to determine whether or not Pc surface components are stable over time in long-term culture. If they are stable, the potential use of axenically cultured Pc as an antigen source will be enhanced. Changes in virulence are often associated with altered surface components. If Pc's surface components change in culture, these organisms will be extremely useful in comparative studies to identify virulence factors in wildtype Pc.